Although RNAi has significant potential to treat cancer by down-regulating oncogenes, this approach shares the classic delivery problem of antisense and gene therapies: negatively charged nucleic acids are poorly transported to their tissue and cellular targets. A promising carrier of siRNA with low toxicity that our lab has developed is composed primarily of histidines and lysines (HK). HK peptides can be tailored to transport siRNA into cells by altering its amino acid sequence; specifically, the repeating pattern of -HHHK- in the terminal branches of HK enables effective siRNA transport in vitro. Our primary goal with HK polymers is to develop a safe and effective delivery method for siRNA in vivo. Addition of Peg and tumor-specific ligands is a cornerstone of therapy for improvement of carriers such as HK. In contrast to many carriers, HK polymers can be pegylated at specific sites, which may augment stability and induce greater endosomal lysis. To test the overall hypothesis that specific pegylation patterns on the most effective HK peptide will markedly increase delivery of siRNA to tumors, the following specific aims are planned. Aim 1 will compare the ability of several unmodified H3K4b derivatives as systemic carriers of Raf-1 siRNA to reduce the size of tumor xenografts. To transport the tumor-inhibitory Raf-1 siRNA systemically to tumor xenografts, we have selected three HK polymers with a predominant repeating amino acid sequence of -HHHK-, an effective sequence for siRNA transport. By varying the lysine core and amino acid sequence of the terminal branches, we hypothesize that H3K4b and its derivatives will differ in their efficacy as in vivo carriers of siRNA targeting Raf-1. This Aim will establish the most effective unmodified HK carrier of siRNA to which pegylation at specific sites will be studied in subsequent aims. Aim 2 will delineate the properties of unmodified and pegylated H3K4b (or derivative) nanoparticles. Several biophysical techniques will be examined, including morphology and stability of pegylated HK:siRNA nanoplexes to correlate their physical properties with the efficacy of siRNA delivery. By comparing HK polymers that have been pegylated at different sites, our hypothesis is that key structural features of the carrier will be identified that will increase stability and augment endosomal lysis. From the knowledge gained in previous aims, Aim 3 will compare a series of pegylated analogs of H3K4b (or derivative) in complex with Raf-1 siRNA for their ability to reduce tumor growth. Based on the structural and mechanistic studies, we hypothesize that specific pegylation patterns on the H3K4b siRNA nanoplex will markedly augment delivery of Raf-1 siRNA to tumor xenografts. Besides increasing the tumor-inhibitory activity of the nanoplex, addition of Peg and a ligand to specific locations on HK should markedly increase specificity of the nanoplex, and consequently reduce toxicity. It is anticipated that this proposal will enhance our understanding of interactions between Peg and HK siRNA polyplexes and that a safe and effective HK carrier will be developed for systemic siRNA-mediated therapy against tumors. PUBLIC HEALTH RELEVANCE: Effective delivery of tumor-inhibitory siRNA is critical for this method of nucleic acid therapeutics to be successful. To achieve this goal, we will utilize nanoparticles composed of modified peptides that interact with these tumor inhibitory siRNA molecules. We will test whether these nanoparticles are able to deliver siRNA to tumors in a mouse model.